The goal of this project is to enhance the means to use mesenchymal stem cells (MSC) for the treatment of peripheral artery disease, particularly in the diabetic patient. MSC transplantation hold great promise as a therapeutic intervention for PAD based on their pluripotency, as well as their efficacy in paracrine delivery of proangiogenic factors. However, we have determined that diabetic MSC manifest greater oxidant stress than healthy (WT) MSC. Diabetic MSC display restricted pluripotency, favoring adipocytic over endothelial differentiation; when transplanted into a WT host, diabetic MSC impair post-ischemic neovascularization and generate fatty infiltration in the ischemic hindlimb. The project hypothesis is that oxidant stress in diabetic MSC restricts their pluripotency and hence their neovascularization capacity. Three aims are proposed to test this hypothesis. Specific Aim 1 will demonstrate that oxidant production is the basis for restricted pluripotency in diabetic MSC, using a reductionist approach in cultured MSC. Exp [1] will determine if Nox4-derived H2O2 drives adipocyte differentiation in diabetic MSC. Exp [2] will determine the role of PPAR3-overexpression in diabetic MSC in generating adipocyte differentiation. Exp [3] will determine the role of uncoupled eNOS in generating oxidant stress in diabetic MSC and evaluate strategies for eNOS recoupling. Exp [4] will determine if deficiencies of the VEGF-Akt-eNOS pathway are the basis for impaired endothelial differentiation in diabetic MSC. Specific Aim 2 will use an in vivo MSC transplant paradigm to demonstrate that antioxidant treatment of diabetic MSCs or the diabetic host improves the efficacy of MSC transplant vis-a-vis post-ischemic neovascularization. Exp [1] will determine if ex vivo treatment of diabetic MSC with N-acetylcysteine (NAC) or other agents with direct or indirect antioxidant properties (resveratrol, rosiglitazone, rosuvastatin) improve their function upon subsequent transplant into a WT host in the setting of hindlimb ischemia. Exp [2] will determine if treatment of the db/db recipient mouse with NAC, or the other agents just noted, improve the outcome of MSC transplant in the setting of hindlimb ischemia. Specific Aim 3 will demonstrate that genetic engineering of MSC to enhance their expression of proangiogenic factors improves their participation in post-ischemic neovascularization. Exp [1] will determine if overexpression of wild type eNOS or constitutively active Akt in MSCs prior to transplant increases eNOS activation and eNOS-derived NO production, and in turn improves their efficacy in the treatment post-ischemic neovascularization. Exp [2] will determine if selection MSC expressing the chemokine receptor CXCR4 increases the homing of these cells to the ischemic hindlimb. If homing is improved, then this CXCR4+ MSC will undergo genetic engineering to maximize their in vivo functional capacity.